Anti-mesothelin immunoconjugates and uses therefor

ABSTRACT

The present invention provides immunoconjugates composed of antibodies, e.g., monoclonal antibodies, or antibody fragments that bind to mesothelin, that are conjugated to cytotoxic agents, e.g., maytansine, or derivatives thereof, and/or co-administered or formulated with one or more additional anti-cancer agents. The immunoconjugates of the invention can be used in the methods of the invention to treat and/or diagnose and/or monitor cancers, solid tumors, recombinant antigen-binding regions and antibodies and functional fragments containing such antigen-binding regions that are specific for the membrane-anchored, 40 kDa mesothelin polypeptide, which which is overexpressed in several tumors, such as pancreatic and ovarian tumors, mesothelioma and lung cancer cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/803,410, filed Jul. 20, 2015, which is acontinuation application of U.S. patent application Ser. No. 13/318,138,filed Jan. 18, 2012, now U.S. Pat. No. 9,084,829, which is the nationalstage of International Application No. PCT/EP2010/002342, filedinternationally on Apr. 16, 2010, which claims the benefit of EuropeanApplication No. 09005909.8, filed Apr. 29, 2009, the disclosures ofwhich are hereby incorporated by reference in their entireties for allpurposes.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:777052009302SubSeqList.txt, date recorded: Apr. 2, 2018, size: 20 KB.

TECHNICAL FIELD

The present invention provides immunoconjugates comprising an antibodyor fragment thereof, having specificity for the mesothelin protein, anda therapeutic agent. Compositions of such immunoconjugates can be usedin treating, preventing, or diagnosing mesothelin-related disorders,e.g. cancer.

BACKGROUND ART

The occurrence of cancer is most commonly associated with aging whereby65% of all new cases of cancer are recorded for patients aged 65 andover. Cancer is the second leading cause of death in the United States,exceeded only by heart disease. Indeed, the American Cancer Society hasestimated that 1 in 4 people will die from cancer in the U.S., assumingcurrent mortality rates remain static. In the U.S. alone, 1,437,180 newcases and 565,650 deaths from cancer are expected in 2008.

Antibody-based therapy is proving very effective in the treatment ofvarious cancers, including solid tumors. For example, HERCEPTIN® hasbeen used successfully to treat breast cancer. Central to thedevelopment of a successful antibody-based therapy is isolation ofantibodies against cell-surface proteins found to be preferentiallyexpressed on tumor cells. The mesothelin precursor polypeptide is aglycophosphatidylinositol (GM-anchored, glycosylated cell surfaceprotein that is proteolytically cleaved to a 30 kDa N-terminal secretedpolypeptide and a 40 kDa, C-terminal polypeptide, which predominantlyoccurs in the membrane-bound, GPI-anchored form (Chang, K. and I.Pastan, Proc. Natl. Acad. Sci. USA, (1996) 93(1):136), and which isnamed mesothelin herein. Mesothelin is preferentially expressed bycertain tumor cells, particularly mesothelioma cells, pancreatic tumorcells and ovarian carcinoma cells, while its expression is limited innormal tissue, making it an attractive target for the development oftumor therapy (Argani, P. et al., Clin. Cancer Res. (2001) 7(12): 3862;Hassan, R., et al., Clin. Cancer Res, (2004) 10(12 Pt 1):3937). Thefunction of mesothelin is unknown, and no apparent reproductive,hematologic, or anatomic abnormalities were observed in mice deficientin mesothelin gene expression (Gera, T. K. and I. Pastan, Mol, Cell.Biol. (2000) 20(8):2902).

Antibody-based, targeted therapy against mesothelin-expressing cancercells has been proposed for the treatment of lung, ovarian andpancreatic cancer. Mab K1 was the first antibody to membrane-boundmesothelin polypeptide which was described (Chang, K., et al., Int. J.Cancer, (1992) 50(3):373). Mab K1 was generated by immunizing mice. Dueto low affinity and poor internalization rates of the antibody, animmunotoxin consisting of Mab K1 linked to a chemically modifiedtruncated form of Pseudomonas exotoxin A was not considered suitable forclinical development (Hassan, R., et al., Immunother. (2000) 23(4):473;Hassan, R., et al., Clin. Cancer Res. (2004) 10(12 Pt 1): 3937).Subsequently, single-chain antibodies with higher affinities weredeveloped, including SS1-(dsFv)-PE38, which showed killing activity oftumor cells in vitro (Hassan, R., et al., Clin. Cancer Res. (2002)8(11): 3520) as well as potency in a murine model of humanmesothelin-expressing tumors (Fan, D., et al., Mol. Cancer Ther. (2002)1(8): 595). These data validate mesothelin as an attractive target todevelop immunotherapy for the treatment of multiple cancers.SS1-(dsFv)-PE38 has been shown to have a fast blood clearance andattempts are being reported to increase the molecular weight bypegylating the fusion protein (Filpula, D., et al., Bioconjugate Chem,(2007) 18(3): 773).

MS-1, MS-2 and MS-3 are mesothelin-binding antibodies which elicitimmune effector activity at the cell surface due to their human IgG1isotype and internalize into mesothelin expressing cells (WO 2006/099141A2). One of these antibodies, the unconjugated, chimeric (mouse/man)IgG1 anti-mesothelin antibody MORAb 009 is currently being tested in aclinical trial for therapeutic effects in the treatment of pancreaticcancer. The postulated mechanism of action of MORAb 009 is triggering ofimmune effector functions such as ADCC and function blocking.

New therapies with improved potency to fight aggressive cancers such asovarian, pancreatic and lung cancer are highly desirable and wouldrepresent an advancement in the art. As such, the present inventiondiscloses new immunoconjugate compositions that are useful in thetreatment, prevention and/or diagnosis of mesothelin-related disorders,e.g. cancer.

SUMMARY OF THE INVENTION

The present invention relates to immunoconjugates comprising ofantibodies, e.g., monoclonal antibodies, or fragments thereof that bindto mesothelin, that are conjugated to cytotoxic agents, e.g.,maytansinoids, or derivatives thereof, and/or are co-administered orformulated with one or more additional anti-cancer agents. Theimmunoconjugates of the invention can be used to treat and/or diagnoseand/or monitor mesothelin-related disorders, e.g. cancer.

It is an object of the invention to provide immunoconjugates comprisingof antibodies, or antigen-binding antibody fragments thereof, orvariants thereof, that are highly selective for the 40 kDa, C-terminalextracellular part of the mesothelin precursor polypeptide, and do bindmesothelin in the presence of cancer antigen 125 (CA125; MUC16), and aneffector moiety. The particular properties of mesothelin antibodies havebeen described in PCT/EP2008/009756, and in one aspect of the invention,the combination of their particular ability to specifically immunoreactwith mesothelin in the presence of CA125 in combination with a cytotoxicagent, e.g. maytansinoid, conjugation provides improved efficacy overfunction blocking antibodies, which compete with CA125 for mesothelinbinding.

In one aspect, the antibodies, or fragments thereof of the invention areIgG antibodies or IgG fragments. The antibodies or fragments can also beIgG1, IgG2a, lgG2b, IgG3, IgM, IgD, IgE, IgA, or IgM antibodies, Fabfragments, F(ab′)2 fragments, scFv fragments, Fv fragments, a diabodies,linear antibodies, single-chain antibodies, biospecific antibodies,multispecific antibodies, or chimeric antibodies (e.g. comprising ahuman antibody scaffold grafted to a human or non-human antibody bindingregion, or a non-human antibody scaffold grafted to a human or non-humanantibody binding region). The chimeric antibodies can include, forexample, antibody scaffold regions from non-human sources, such as, forexample, cow, mouse, llama, camel, or rabbit. Further information on theengineering of antibodies can be found in the literature, for example,Holliger and Hudson, Nature Biotechnology, (gy, (September 2005) 23:1126-1136, which is incorporated herein by reference. The aforementionedfragments can be obtained from an immunoglobulin or produced by asuitable means, e.g. recombinant expression, in a fragment form.

The antibodies or antibody fragments of the invention can also behumanized, wherein the CDR sequences or regions (e.g. CDR1, CDR2, CDR3)can be non-human, e.g. murine.

The antibodies or antibody fragments of the invention, or compositionsincluding the antibodies or fragments, can include a cytoxic agent thatis conjugated to an antibody or fragment thereof. In one aspect, thecytotoxic agent is a maytansinoid or a dertivative thereof, however,other cytoxic agents are also provided, which can include, for example,and other cytotoxic agents, e.g., aplidin, auristatin, azaribine,anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine,celebrex, chlorambucil, cisplatin, irinotecan (CPT-I 1), SN-38,carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactirromycin, daunomycin glucuronide, daunorubicin,dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3°,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone,procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin,tamoxifen, taxanes, taxol, testosterone propionate, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade,vinblastine, vinorelbine, vincristine, ricin, abrin, ribomiclease,onconase, rapLRI, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, or combinations thereof.Any of the cytoxic agents can also include functional analogs thereof orderivatives thereof.

In another aspect, the present invention provides immunoconjugates inwhich the cytotoxic agent is non-immunogenic, i.e. does not increase theimmunogenicity of the parental antibody by contributing human ormammalian B cell epitopes or T cell epitopes to a drug formulation.

The compositions of the invention can include in addition to theantibodies and fragments (with or without the aforementioned conjugatedcytoxic agents) various anticancer agents, which can include, forexample, bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate,erlotinib (Tarceva), etoposide, finasteride (Proscar), flutamide(Eulexin), gemcitabine (Gemzar), genitinib (Lrresa), goserelin acetate(Zoladex), granisetron (Kytril), imatinib (Gleevec), irinotecan(Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol),pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen), porfimersodium (Photofrin), interleukin-2 (Proleukin), rituximab (Rituxan),topotecan (Hycamtin), trastuzumab (Herceptin), Triapine, vincristine,and vinorelbine tartrate (Navelbine), or therapeutic antibodies orfragments thereof, or anti-angiogenic agent, such as, for example,angiostatin, bevacizumab (Avastin®), sorafenib (Nexavar®), baculostatin,canstatin, maspin, anti-VEGF antibodies or peptides, anti-placentalgrowth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Fit-1antibodies or peptides, laminin peptides, fibronectin peptides,plasminogen activator inhibitors, tissue metalloproteinase inhibitors,interferons, interleukin 12, IP-IO, Gro-β thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CMIOI, Marimastat, pentosan polysulphate, angiopoietin 2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470,platelet factor 4 or minocycline.

The present invention further provides in another aspect a method fortreating a mesothelin-related disorder by administering atherapeutically effective amount of the immunoconjugates of theinvention, or the compositions of the invention which include theimmunoconjugates of the Invention. The mesothelin-related disorder caninclude, for example, cancer, such as, a solid tumor cancer. The solidtumor can be in or originating from the ovary, pancreas, respiratorytract, lung, colon, stomach, esophagus, cervix, liver, breast, head, andneck.

These and other embodiments are disclosed or are obvious from andencompassed by, the following detailed description.

DESCRIPTION OF THE FIGURES

FIGS. 1A-B show anti-tumor efficacy of anti mesothelin-immunoconjugateMF-T-SPDB-DM4 on mesothelin-transfected human pancreas carcinoma cellsin a mesothelin transfected xenograft model (FIG. 1A) as well as innon-transfected control tumors (FIG. 1B).

FIG. 2 shows anti-tumor efficacy of anti-mesothelin immunoconjugateswith stable and cleavable, as well as polar and nonpolar linkers in aHelaMATU xenograft model with carcinoma cells expressing mesothelinendogenously.

FIGS. 3A-B show anti-tumor efficacy of anti-mesothelin immunoconjugateswith stable and cleavable, as well as polar and nonpolar linkers in amesothelin transfected xenograft model (FIG. 3A) as well as innon-transfected control tumors (FIG. 3B).

FIG. 4 shows an example of a dose response cur NIF-T-SPDP-DM4 toxicityon mesothelin positive HelaMatu cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novelimmunoconjugates that are specific to or have a high affinity formesothelin and can deliver a therapeutic benefit to a subject. Theimmunoconjugates of the invention can be used in many contexts, whichare more fully described herein. It is to be understood that presentinvention as described herein is not to be limited to the particulardetails set forth herein regarding any aspect of the invention,including, anti-mesothelin antibodies, immunoconjugates, methods oftreatment, protocols, cell lines, animal species or genera, constructs,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. The following references, however, canprovide one of skill in the art to which this invention pertains with ageneral definition of many of the terms used in this invention, and canbe referenced and used so long as such definitions are consistent themeaning commonly understood in the art. Such references include, but arenot limited to, Singleton et ah, Dictionary of Microbiology andMolecular Biology (2d ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); Hale & Marham, The Harper CollinsDictionary of Biology (1991); and Lackie et al., The Dictionary of Cell& Molecular Biology (3d ed. 1999); and Cellular and MolecularImmunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W. B. SaundersCompany. Any additional technical resources available to the person ofordinary skill in the art providing definitions of terms used hereinhaving the meaning commonly understood in the art can be consulted. Forthe purposes of the present invention, the following terms are furtherdefined. Additional terms are defined elsewhere in the description. Asused herein and in the appended claims, the singular forms “a,” “and,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a gene” is a reference toone or more genes and includes equivalents thereof known to thoseskilled in the art, and so forth.

As used herein, the term “antibody” includes immunoglobulin molecules(e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY, and/or anyclass, including, IgG1, IgG2, IgG3, IgG4, IgA1 and Ig A2) isolated fromnature or prepared by recombinant means. Antibodies also are meant toencompass antigen-binding antibody fragments, such as Fab, F(ab′)2, scFv(single chain Fvs), Fv, single chain antibodies, diabodies,disulfide-linked Fvs (sdFv), and fragments comprising a VL or VH domain,which are prepared from intact immunoglobulins or prepared byrecombinant means.

The antibodies and/or antigen-binding antibody fragments of the presentinvention may be monospecific (e.g. monoclonal), bispecific, trispecificor of greater multi specificity. Multispecific antibodies may bespecific for different epitopes of an antigen or may be specific forepitopes of more than one antigen. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J.Immunol. 147:60 69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;5,573,920; 5,601,819; Kostelny et al., 1992, J. ImmunoL 148:1547 1553,each of which are incorporated herein by reference.

Antigen-binding antibody fragments may comprise the variable region(s)alone or in combination with the entirety or a portion of the following:hinge region, CHI, CH2, CH3 and CL domains. Also included in theinvention are antigen-binding antibody fragments also comprising anycombination of variable region(s) with a hinge region, CHI, CH2, CH3 andCL domain.

Preferably, the antibodies or antigen-binding antibody fragments arehuman, humanized, murine (e.g., mouse and rat), donkey, sheep, rabbit,goat, guinea pig, camelid, horse, or chicken. As used herein, “human”antibodies include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries, from human B cells, or from animals transgenic for one ormore human immunoglobulin, as described infra and, for example in U.S.Pat. No. 5,939,598 by Kucherlapati et al. The term antibody also extendsto other protein scaffolds that are able to orient antibody CDR insertsinto the same active binding conformation as that found in naturalantibodies such that binding of the target antigen observed with thesechimeric proteins is maintained relative to the binding activity of thenatural antibody from which the CDRs were derived. As used herein, theterm “humanized” forms of non-human (e.g., murine) antibodies arechimeric antibodies which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which hypervariable regionresidues (e.g. the complementarity determining regions “CDR”) of therecipient are replaced by hypervariable region residues (CDRs) from anon-human species (donor antibody) such as mouse, rat, rabbit, ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin may be replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Suchmodifications are made to further refine antibody performance. Ingeneral, the humanized antibody may comprise substantially all of atleast one or typically two variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also may comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. For areview, see Jones, et al., (Nature 321:522-525, 1986): Reichmann, etal., (Nature 332:323-329, 1988); and Presta, (Curr. Op. Struct. Biol.2:593-596, 1992). The preparation of humanized antibodies can be foundin U.S. Pat. Nos. 7,049,135, 6,828,422, 6,753,136, 6,706,484, 6,696,248,6,692,935, 6,667,150, 6,653,068, 6,300,064, 6,294,353, and 5,514,548,each of which are incorporated herein in their entireties.

As used herein, the term “single-chain Fv” or “sFv” antibody fragmentscomprise the VH and VL domains of an antibody, wherein these domains arepresent in a single polypeptide chain. Generally, the Fv polypeptidefurther comprises a polypeptide linker between the VH and VL domainswhich enables the sFv to form the desired structure for antigen binding.For a review, see Pluckthun (The Pharmacology of Monoclonal Antibodies.Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315, 1994), which is incorporated herein in its entirety byreference.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger, et al., (Proc.Natl. Acad. Sci. USA 90:6444-6448, 1993), each of which are incorporatedby reference.

The expression “linear antibodies” refers to the antibodies described inthe art, for example, in Zapata, et al., (Protein Eng. 8(10): 1057-1062,1995), which is incorporated by reference. Briefly, such antibodiescomprise a pair of tandem Fd segments (VH-CHI-VH-CHI) which form a pairof antigen binding regions. Linear antibodies can be bispecific ormonospecific.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, thatis, individual antibodies 10 comprising an identical population exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, that is, directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler, et al., (Nature256:495, 1975), or may be made by recombinant DNA methods {see, e.g.,U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolatedfrom phage antibody libraries using the techniques described in, forexample, Clackson, et al., (Nature 352:624-628,1991) and Marks, et al.,(J. Mol. Biol. 222:581-597, 1991).

The monoclonal antibodies herein also include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984, each of which areincorporated by reference).

As used herein, the terms “biological sample” or “patient sample” asused herein, refers to a sample obtained from an organism or fromcomponents (e.g., cells) of an organism. The sample may be of anybiological tissue or fluid. The sample may be a “clinical sample” whichis a sample derived from a patient. Such samples include, but are notlimited to, sputum, blood, serum, plasma, blood cells (e.g., whitecells), tissue samples, biopsy samples, urine, peritoneal fluid, andpleural fluid, saliva, semen, breast exudate, cerebrospinal fluid,tears, mucous, lymph, cytosols, ascites, amniotic fluid, bladder washes,and bronchioalveolar lavages or cells therefrom, among other body fluidsamples. The patient samples may be fresh or frozen, and may be treatedwith heparin, citrate, or EDTA. Biological samples may also includesections of tissues such as frozen sections taken for histologicalpurposes.

The term “cancer” includes, but is not limited to, solid tumors, such ascancers of the pancreas, breast, respiratory tract, brain, reproductiveorgans, digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid, and their distant metastases. The term alsoincludes sarcomas, lymphomas, leukemias, and plasma cell myelomas.

Tumors of the respiratory tract include, but are not limited to,small-cell and non-small-cell lung carcinoma, as well as bronchialadenoma and pleuropulmonary blastoma. Tumors of breast include, but arenot limited to, invasive ductal carcinoma, invasive lobular carcinoma,ductal carcinoma in situ, and lobular carcinoma in situ. Tumors of braininclude, but are not limited to, brain stem and hypophtalmic glioma,cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, aswell as neuroectodermal and pineal tumor. Tumors of the malereproductive organs include, but are not limited to, prostate andtesticular cancer. Tumors of the female reproductive organs include, butare not limited to, endometrial, cervical, ovarian, vaginal, and vulvarcancer, as well as sarcoma of the uterus. Tumors of the digestive tractinclude, but are not limited to, anal, colon, colorectal, esophageal,gallbladder, gastric, pancreatic, rectal, small-intestine, and salivarygland cancers. Tumors of the urinary tract include, but are not limitedto, bladder, penile, kidney, renal pelvis, ureter, and urethral cancers.Eye cancers include, but are not limited to, intraocular melanoma andretinoblastoma. Tumors of liver include, but are not limited to,hepatocellular carcinoma (liver cell carcinomas with or withoutfibrolamellar variant), cholangiocarcinoma (intrahepatic bile ductcarcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancersinclude, but are not limited to, squamous cell carcinoma, Kaposi'ssarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanomaskin cancer. Head-and-neck cancers include, but are not limited to,laryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and lipand oral cavity cancer. Lymphomas include, but are not limited to,AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-celllymphoma, Hodgkin's disease, and lymphoma of the central nervous system.Sarcomas include, but are not limited to, sarcoma of the soft tissue,osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, andrhabdomyosarcoma. Leukemias include, but are not limited to, acutemyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, and hairy cell leukemia.

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen, e.g. mesothelin protein, to which theantibody binds through an antigenic binding site. Determinants orantigenic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

The term “specifically immunoreactive” refers to a binding reactionbetween an antibody and a protein, compound, or antigen, having anepitope recognized by the antigenic binding site of the antibody. Thisbinding reaction is determinative of the presence of a protein, antigenor epitope having the recognized epitope amongst the 10 presence of aheterogeneous population of proteins and other biologies. In the contextof an immunoassay, specifically immunoreactive antibodies can bind to aprotein having the recognized epitope and bind, if at all, to adetectably lesser degree to other proteins lacking the epitope which arepresent in the sample. In an in vivo context, “specificallyimmunoreactive” can refer to the conditions under which in an animalforms an immune response against a vaccine or antigen, e.g. a humoralresponse to the antigen (the production of antibodies, against avaccine, protein, compound, or antigen presented thereto underimmunologically reactive conditions) or a cell-mediated (also herein as“cellular immune response”, i.e, a response mediated by T lymphocytesagainst the vaccine, protein, compound or antigen presented thereto). Asused herein, the term “immunologically reactive conditions” is used inthe context of an immunoassay or an in vitro reaction wherein thephysical conditions of the reaction, including, for example, thetemperature, salt concentration, pH, reagents and their concentrations,and the concentrations of antigen and cognate antibody that isspecifically immunoreactive to the antigen, are provided or adjusted toallow binding of the cognate antibody to the antigen. Immunologicallyreactive conditions are dependent upon the format of the antibodybinding reaction and, typically are those utilized in immunoassayprotocols. See Harlow and Lane (1988) Antibodies: A Laboratory Manual,Cold Spring Harbor Publications, New York, for a description ofimmunoassay formats and conditions. The term “patient” or “subject” asused herein includes mammals (e.g., humans and animals).

As used herein, the term ‘invariant binding’ of a particular antibody tomesothelin refers to its ability to bind to mesothelin on a broad rangeof mesothelin-expressing cancer cell lines which express different formsof mesothelin. Invariant binding may be caused by, but is not restrictedto, the fact that antibodies, or antigen-binding antibody fragmentsthereof, or variants thereof, recognize an epitope of mesothelin that isnot masked by another extracellular antigen, such as cancer antigen 125(CA125), which interacts with mesothelin. For invariantly bindingantibodies, EC50 values determined by FACS titration on two distinctcancer cell lines might differ no more than 10 fold, or, preferably,5fold, and most preferably between 1 and 3 fold.

-   -   As used herein, the term “immunoconjugate” refers to a conjugate        molecule comprising at least one antibody or an antigen-binding        fragment thereof, bound to a cytotoxic agent, e.g., a        maytansinoid or a derivative thereof, preferably via a suitable        linking group or a precursor thereof.        Immunoconjugates of the Invention

The present invention relates to methods to inhibit growth ofmesothelin-positive cancer cells and the progression of neoplasticdisease by providing anti-mesothelin immunoconjugates. The antibodymoiety of the provided immunoconjugates are specifically immunoreactiveto the 40 kDa, C-terminal domain of the mesothelin precursor polypeptide(SEQ ID NO 36), which is named ‘mesothelin’ herein.

In one aspect of the invention, the antibodies, antigen-binding antibodyfragments, and variants of the antibodies and fragments of the inventionhave been described in PCT/EP2008/009756 and are comprised of a lightchain variable region and a heavy chain variable region. Variants of theantibodies or antigen-binding antibody fragments contemplated in theinvention are molecules in which the binding activity of the antibody orantigen-binding antibody fragment for mesothelin is maintained.

The present invention also relates to immunoconjugates composed ofanti-mesothelin antibodies, antigen-binding antibody fragments, andvariants of the antibodies and fragments of the invention different tothose which have been described in (PCT/EP2008/009756), and linked to achemotherapeutic agent, e.g. maytansinoids, or derivatives thereof.

Maytansinoids that can be used in the present invention are well knownin the art and can be isolated from natural sources according to knownmethods or prepared synthetically according to known methods.

Examples of suitable maytansinoids include maytansinol and maytansinolanalogues. Examples of suitable maytansinol analogues include thosehaving a modified aromatic ring and those having modifications at otherpositions.

Specific examples of suitable analogues of maytansinol having a modifiedaromatic ring include:

(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reductionof ansamitocin P2);

(2) C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction ofmaytansinol with H2S or P2S5);

(2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by theconversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudiflora);

(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (preparedby the demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

The synthesis of thiol-containing maytansinoids useful in the presentinvention is fully disclosed in U.S. Pat. Nos. 5,208,020, 5,416,064, and7,276,497.

Maytansinoids with a thiol moiety at the C-3 position, the C-14position, the C-15 position or the C-20 position are all expected to beuseful. The C-3 position is preferred and the C-3 position ofmaytansinol is especially preferred. Also preferred are anN-methyl-alanine-containing C-3 thiol moiety maytansinoid, and anN-methyl-cysteine-containing C-3 thiol moiety maytansinoid, andanalogues of each. Preferred maytansinoids are those described in U.S.Pat. Nos. 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821;RE39,151 and 7,276,497, each of which is incorporated herein in theirentirety by reference. In a preferred embodiment, the esterifiedmaytansinol is selected fromN2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1, CAS Reg. No.139504-50-0), N2′-deacetyl-N2′-(4-mercapto-1-oxopentyl)-maytansine (DM3,CAS Reg. No 796073-54-6), andN2′-deacetyl-N2′-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4 CASReg. No. 796073-69-3).

Throughout this document, reference is made to the followingrepresentative antibodies of the invention: “MF-J”, “MOR06640”,“MF-226”, and “MF-T”. MF-J represents an antibody having a variableheavy region corresponding to SEQ ID NO: 28 (DNA)/SEQ ID NO: 20(protein) and a variable light region corresponding to SEQ ID NO: 32(DNA)/SEQ ID NO: 24 (protein). MOR 06640 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 29 (DNA)/SEQ ID NO: 21(protein) and a variable light region corresponding to SEQ ID NO: 33(DNA)/SEQ ID NO: 25 (protein). MF-226 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 30 (DNA)/SEQ ID NO: 22(protein) and a variable light region corresponding to SEQ ID NO: 34(DNA)/SEQ ID NO: 26 (protein). MF-T represents an antibody having avariable heavy region corresponding to SEQ ID NO: 31 (DNA)/SEQ ID NO: 23(protein) and a variable light region corresponding to SEQ ID NO: 35(DNA)/SEQ ID NO: 27 (protein). The invention is not limited to theseantibodies which are used here as are examples. Other usefull antibodiesare disclosed for example in PCT/EP2008/009756.

In one aspect, the invention provides immunoconjugates that arespecifically immunoreactive to mesothelin in the presence of cancerantigen 125 (CA 125/MUC 16), and therefore efficiently target cancercells expressing both mesothelin and CA125, e.g. OVCAR-3 cells.

-   -   In other aspects the invention provides immunoconjugates which        are specifically immunoreactive to one or more amino acids of        the epitopes of antibodies MOR 06640 or MF-T. In certain aspects        said immunoconjugates are specifically immunoreactive to at        least to two, at least three, at least four, at least five or at        least six amino acids of the epitopes of antibodies MOR 06640 or        MF-T. In certain aspects the immunoconjugates of the present        invention are specifically immunoreactive to one or more amino        acids of the epitope recognized by the antibody MOR 06640. In        alternative aspects the antibodies of the present invention are        specifically immunoreactive to one or more amino acids of the        epitope recognized by the antibody MF-T.

In another aspect, the invention provides immunoconjugates having anantigen-binding region that is specifically immunoreactive to or has ahigh affinity for one or more regions of mesothelin, whose amino acidsequence is depicted by SEQ ID NO: 36. An immunoconjugate is said tohave a “high affinity” for an antigen if the affinity measurement is atleast 100 nM (monovalent affinity of Fab fragment). An inventiveimmunoconjugate preferably can be specifically immunoreactive tomesothelin with an affinity of less than about 100 nM, more preferablyless than about 60 nM, and still more preferably less than about 30 nM.Further preferred are antibodies that bind to mesothelin with anaffinity of less than about 10 nM, and more preferably less than about 3nM. For instance, the affinity of an antibody of the invention againstmesothelin may be about 9.1 nM or 0.9 nM (monovalent affinity of IgG1format).

Methods of Use

The term “treatment” includes any process, action, application, therapy,or the like, wherein a subject (or patient), including a human being, isprovided medical aid with the object of improving the subject'scondition, directly or indirectly, or slowing the progression of acondition or -disorder in the subject, or ameliorating at least onesymptom of the disease or disorder under treatment.

The term “combination therapy” or “co-therapy” means the administrationof two or more therapeutic agents to treat a disease, condition, and/ordisorder. Such administration encompasses co-administration of two ormore therapeutic agents in a substantially simultaneous manner, such asin a single capsule having a fixed ratio of active ingredients or inmultiple, separate capsules for each inhibitor agent. In addition, suchadministration encompasses use of each type of therapeutic agent in asequential manner. The order of administration of two or moresequentially co-administered therapeutic agents is not limited. Thephrase “therapeutically effective amount” means the amount of each agentadministered that will achieve the goal of improvement in a disease,condition, and/or disorder severity, and/or symptom thereof, whileavoiding or minimizing adverse side effects associated with the giventherapeutic treatment.

The term “pharmaceutically acceptable” means that the subject item isappropriate for use in a pharmaceutical product.

The immunoconjugates of this invention are expected to be valuable astherapeutic agents. Accordingly, an embodiment of this inventionincludes a method of treating the various conditions in a patient(including mammals) which comprises administering to said patient acomposition containing an amount of an immunoconjugate of the inventionthat is effective in treating the target condition.

The immunoconjugates of the present invention may be used in thetreatment or prevention of diseases and/or behaviors that are associatedwith the mesothelin protein. These diseases and/or behaviors include,for example, cancer, such as, carcinomas of the pancreas, ovary,stomach, esophagus, cervix, colon, liver, respiratory tract, and lung.The present invention also relates to methods of ameliorating symptomsof a disorder in which mesothelin is elevated or otherwise abnormallyexpressed. These disorders include, without limitation, carcinomas ofthe pancreas, ovary, stomach, esophagus, cervix, colon, liver,respiratory tract, and lung (see, e.g., (Liao, Cancer Res. 57:2827-2831,1997; Turner, Hurn. Pathol. 28:740-744, 1997; Liao, et al., Am. J.Pathol. 145:598-609, 1994; Saarnio, et al., Am. J. Pathol. 153:279-285,1998; Vermylen, et al., Eur. Respir. J. 14:806-811, 1999). In oneembodiment of the invention, a therapeutically effective dose of animmunoconjugate of the invention is administered to a patient having adisorder in which mesothelin is elevated.

Immunoconjugates of the present invention may be administered alone orin combination with one or more additional therapeutic agents.Combination therapy includes administration of a single pharmaceuticaldosage formulation which contains an immunoconjugate of the presentinvention and one or more additional therapeutic agents, as well asadministration of the immunoconjugate of the present invention and eachadditional therapeutic agents in its own separate pharmaceutical dosageformulation. For example, an immunoconjugate of the present inventionand a therapeutic agent may be administered to the patient together in asingle oral dosage composition or each agent may be administered inseparate oral dosage formulations. Where separate dosage formulationsare used, the immunoconjugate of the present invention and one or moreadditional therapeutic agents may be administered at essentially thesame time (e.g., concurrently) or at separately staggered times (e.g.,sequentially). The order of administration of the agents is not limited.

For example, in one aspect, co-administration of an anti-mesothelinimmunoconjugate of the invention together with one or more anti-canceragents to potentiate the effect of either the anti-mesothelinimmunoconjugate or the anti-cancer agent(s) or both is contemplated foruse in treating mesothelin-related disorders, such as, cancer. Suchcombination-therapies may also be used to prevent cancer, prevent therecurrence of cancer, prevent the spread or metastasis of a cancer, orreduce or ameliorate the symptoms associated with cancer.

The one or more anti-cancer agents can include any known and suitablecompound in the art, such as, for example, chemoagents, otherimmunotherapeutics, cancer vaccines, anti-angiogenic agents, cytokines,hormone therapies, gene therapies, and radiotherapies. A chemoagent (or“anti-cancer agent” or “anti-tumor agent” or “cancer therapeutic”)refers to any molecule or compound that assists in the treatment of acancer. Examples of chemoagents contemplated by the present inventioninclude, but are not limited to, cytosine arabinoside, taxoids (e.g.,paclitaxel, docetaxel), anti-tubulin agents (e.g., paclitaxel,docetaxel, epothilone B, or its analogues), macrolides (e.g., rhizoxin)cisplatin, carboplatin, adriamycin, tenoposide, mitozantron,discodermolide, eleutherobine, 2-chlorodeoxyadenosine, alkylating agents(e.g., cyclophosphamide, mechlorethamine, thioepa, chlorambucil,melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide,busulfan, dibromomannitol, streptozotocin, mitomycin C, andcis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa),antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,mithramycin, anthramycin), antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, flavopiridol,5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozolamide),asparaginase, Bacillus Calmette and Guerin, diphtheria toxin,hexamethylmelamine, hydroxyurea, LYSODREN®, nucleoside analogues, plantalkaloids (e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan(CAMPTOSAR, CPT-II), vincristine, vinca alkyloids such as vinblastine),podophyllotoxin (including derivatives such as epipodophyllotoxin, VP-16(etoposide), VM-26 (teniposide)), cytochalasin B, colchine, gramicidinD, ethidium bromide, emetine, mitomycin, procarbazine, mechlorethamine,anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin,doxorubicin liposomal), dihydroxyanthracindione, mitoxantrone,mithramycin, actinomycin D, procaine, tetracaine, lidocaine,propranolol, puromycin, anti-mitotic agents, abrin, ricin A, pseudomonasexotoxin, nerve growth factor, platelet derived growth factor, tissueplasminogen activator, aldesleukin, allutamine, anastrozle,bicalutamide, biaomycin, busulfan, capecitabine, carboplain,chlorahusil, cladribine, cylarahine, daclinomycin, estramusine,floxuridhe, gamcitabine, gosereine, idarubicin, itosfamide, lauprolideacetate, levamisole, lomusline, mechlorethamine, magestrol, acetate,mercaptopurino, mesna, mitolanc, pegaspergase, pentostatin, picamyein,riuxlmab, campath-1, straplozocin, thioguanine, tretinoin, vinorelbine,or any fragments, family members, or derivatives thereof, includingpharmaceutically acceptable salts thereof. Compositions comprising oneor more chemoagents (e.g., FLAG, CHOP) are also contemplated by thepresent invention. FLAG comprises fludarabine, cytosine arabinoside(Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine,doxorubicin, and prednisone.

The chemoagent can be an anti-angiogenic agent, such as, for example,angiostatin, bevacizumab (Avastin®), sorafenib (Nexavar®), baculostatin,canstatin, maspin, anti-VEGF antibodies or peptides, anti-placentalgrowth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Fit-1antibodies or peptides, laminin peptides, fibronectin peptides,plasminogen activator inhibitors, tissue metalloproteinase inhibitors,interferons, interleukin 12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CMIOI, Marimastat, pentosan polysulphate, angiopoietin 2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470,platelet factor 4 or minocycline.

In one aspect, said chemoagent is gemcitabine at a dose ranging from 100to 1000 mg/m2/cycle. In one embodiment, said chemoagent is dacarbazineat a dose ranging from 200 to 4000 mg/m2 cycle. In another aspect, saiddose ranges from 700 to 1000 mg/m2/cycle. In yet another aspect, saidchemoagent is fludarabine at a dose ranging from 25 to 50 mg/m2/cycle.In another aspect, said chemoagent is cytosine arabinoside (Ara-C) at adose ranging from 200 to 2000 mg/m2/cycle. In still another aspect, saidchemoagent is docetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle.In yet another aspect, said chemoagent is paclitaxel at a dose rangingfrom 5 to 15 mg/kg/cycle. In a further aspect, said chemoagent iscisplatin at a dose ranging from 5 to 20 mg/kg/cycle. In a still furtheraspect, said chemoagent is 5-fiuorouracil at a dose ranging from 5 to 20mg/kg/cycle. In another aspect, said chemoagent is doxorubicin at a doseranging from 2 to 8 mg/kg/cycle. In yet a further aspect, saidchemoagent is epipodophyllotoxin at a dose ranging from 40 to 160mg/kg/cycle. In yet another aspect, said chemoagent is cyclophosphamideat a dose ranging from 50 to 200 mg/kg/cycle. In a further aspect, saidchemoagent is irinotecan at a dose ranging from 50 to 150 mg/m2/cycle.In a still further aspect, said chemoagent is vinblastine at a doseranging from 3.7 to 18.5 mg/m2/cycle. In another aspect, said chemoagentis vincristine at a dose ranging from 0.7 to 2 mg/m2/cycle. In oneaspect, said chemoagent is methotrexate at a dose ranging from 3.3 to1000 mg/m/cycle.

In another aspect, the anti-mesothelin immunoconjugates of the presentinvention are administered in combination with one or moreimmunotherapeutic agents, such as antibodies or immunomodulators, whichinclude, but are not limited to, Herceptin®, Retuxan®, OvaRex, Panorex,BEC2, IMC-C225, Vitaxin, Campath I/H, Smart MI95, LymphoCide, Smart I D10, and Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.

The invention also contemplates administering the anti-mesothelinimmunoconjugates of the present invention with one or moreanti-angiogenic agents, which includes, but is not limited to,angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine ProteaseInhibitor) anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminalproteolytic fragments of fibronectin, 16 kDa proteolytic fragment ofprolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a β-aminoacid peptide corresponding to a fragment of platelet factor-4 (Maione etal., 1990, Cancer Res. 51:2077), a 14-amino acid peptide correspondingto a fragment of collagen 1 (Tolma et al., 1993, J. Cell Biol. 122:497),a 19 amino acid peptide corresponding to a fragment of Thrombospondin I(Tolsma et al., 1993, J. Cell Biol. 122:497), a 20-amino acid peptidecorresponding to a fragment of SPARC (Sage et al., 1995, J. Cell.Biochem. 57: 1329-), or any fragments, family members, or derivativesthereof, including pharmaceutically acceptable salts thereof. Otherpeptides that inhibit angiogenesis and correspond to fragments oflaminin, fibronectin, procollagen, and EGF have also been described (Seethe review by Cao, 1998, Frog. Mol. Subcell. Biol. 20:161). Monoclonalantibodies and cyclic pentapeptides, which block certain integrins thatbind RGD proteins (i.e., possess the peptide motif Arg-Gly-Asp), havebeen demonstrated to have anti-vascularization activities (Brooks etal., 1994, Science 264:569; Hammes et al., 1996, Nature Medicine 2:529).Moreover, inhibition of the urokinase plasminogen activator receptor byantagonists inhibits angiogenesis, tumor growth and metastasis (Min etal., 1996, Cancer Res. 56:2428-33; Crowley et aL, 1993, Proc Natl Acad.Sci. USA 90:5021). Use of such anti-angiogenic agents is alsocontemplated by the present invention.

In another-aspect, the anti-mesothelin immunoconjugates of the presentinvention are administered in combination with a regimen of radiation.

The anti-mesothelin immunoconjugates of the present invention can alsobe administered in combination with one or more cytokines, whichincludes, but is not limited to, lymphokines, tumor necrosis factors,tumor necrosis factor-like cytokines, lymphotoxin-α, lymphotoxin-β,interferon-β, macrophage inflammatory proteins, granulocyte monocytecolony stimulating factor, interleukins (including, but not limited to,interleukin-1, interleukin-2, interleukin-6, interleukin-12,interleukin-15, interleukin-18), OX40, CD27, CD30, CD40 or CD 137ligands, Fas-Pas ligand, 4-IBBL, endothelial monocyte activating proteinor any fragments, family members, or derivatives thereof, includingpharmaceutically acceptable salts thereof.

The anti-mesothelin immunoconjugates of the present invention can alsobe administered in combination with a cancer vaccine, examples of whichinclude, but are not limited to, autologous cells or tissues,non-autologous cells or tissues, carcinoembryonic antigen,alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine,melanocyte lineage proteins (e.g., gpIOO, MART-1/MelanA, TRP-1 (gp75),tyrosinase, widely shared tumor-associated, including tumor-specific,antigens (e.g., BAGE, GAGE-I, GAGE-2, MAGE-I, MAGE-3,N-acetylglucosaminyltransferase-V, pI5), mutated antigens that aretumor-associated (β-catenin, CDK4), nonmelanoma antigens (e.g.,HER-2/neu (breast and ovarian carcinoma), human 5 papillomavirus-E6, E7(cervical carcinoma), MUC-1 (breast, ovarian and pancreatic carcinoma).For human tumor antigens recognized by T-cells, see generally Robbinsand Kawakami, 1996, Curr. Opin. Immunol. 8:628. Cancer vaccines may ormay not be purified preparations.

In yet another embodiment, the anti-mesothelin immunoconjugates of thepresent invention are used in association with a hormonal treatment.Hormonal therapeutic treatments comprise hormonal agonists, hormonalantagonists (e.g., flutamide, tamoxifen, leuprolide acetate (LUPRON),LH-RH antagonists), inhibitors of hormone biosynthesis and processing,and steroids (e.g., dexamethasone, retinoids, betamethasone, Cortisol,cortisone, prednisone, dehydrotestosterone, glucocorticoids,mineralocorticoids, estrogen, testosterone, progestins), antigestagens(e.g., mifepristone, onapristone), and antiandrogens (e.g., cyproteroneacetate). The anti-mesothelin immunoconjugates of the invention can beused in combination with, e.g. co-administered with, an anti-MDR(multidrug resistance) phenotype agent. Many human cancers intrinsicallyexpress or spontaneously develop resistance to several classes ofanticancer drugs at the same time, notwithstanding that each of the drugclasses have different structures and mechanisms of action. Thisphenomenon, which can be mimicked in cultured mammalian cells, isgenerally referred to as multidrug resistance (“MDR”) or the multidrugresistance phenotype. The MDR phenotype presents significant obstaclesto the successful chemotherapeutic treatments for cancers in humanpatients. Resistance of malignant tumors to multiple chemotherapeuticagents is a major cause of treatment failure (Wittes et al., CancerTreat. Rep. 70:105 (1986); Bradley, G. et al., Biochim. Biophys. Acta948:87 (1988); Griswald, D. P. et al., Cancer Treat. Rep. 65(S2):51(1981); Osteer, R. T. (ed.), Cancer Manual, (1990)). Tumors initiallysensitive to cytotoxic agents often recur or become refractory tomultiple chemotherapeutic drugs (Riordan et al., Pharmacol. Ther. 28:51(1985); Gottesman et al., Trends Pharmacol. Sci. 9:54 (1988); Moscow etal., J. Natl. Cancer Inst. 80:14 (1988); Croop, J. M. et al., J. Clin.Invest. 81:1303 (1988)). Cells or tissues obtained from tumors and grownin the presence of a selecting cytotoxic drug can result incross-resistance to other drugs in that class as well as other classesof drugs including, but not limited to, anthracyclines, Vinca alkaloids,and epipodophyllotoxins (Riordan et al., Pharmacol. Ther. 28:51 (1985);Gottesman et al., J. Biol. Chem. 263:12163 (1988)). Thus, acquiredresistance to a single drug results in simultaneous resistance to adiverse group of drugs that are structurally and functionally unrelated.Such resistance can be a problem for both solid-form and liquid-formtumors (e.g. blood or lymph-based cancers).

One major mechanism of multidrug resistance in mammalian cells involvesthe increased expression of the 170 kDa plasma membrane glycoproteinpump system (Juranka et al., FASEB J 3:2583 (1989); Bradley, G. et al.,Blochem. Biophys. Acta 948:87 (1988)). The gene encoding this pumpsystem, sometimes referred to as a multidrug transporter, has beencloned from cultured human cells and is generally referred to as mdrl.This gene is expressed in several classes of normal tissues, butphysiological substrates transported for the mdrl gene product in thesetissues have not been identified. The MDRI product is a member of theABC Transporter Protein superfamily, a group of proteins havingenergy-dependent export function. The protein product of the mdrl gene,generally known as P-glycoprotein (“P-170”, “P-gp”), is a 170 kDatrans-plasma membrane protein that constitutes the aforementionedenergy-dependent efflux pump. Expression of P-gp on the cell surface issufficient to render cells resistant to multiple cytotoxic drugs,including many anti-cancer agents. P-gp-mediated MDR appears to be animportant clinical component of tumor resistance in tumors of differenttypes, and mdrl gene expression correlates with resistance tochemotherapy in different types of cancer. The nucleotide sequence ofthe mdrl gene (Gros, P. et al., Cell 47:371 (1986); Chen, C. et al.,Cell 47:381 (1986)) indicates that it encodes a polypeptide similar oridentical to P-glycoprotein and that these are members of the highlyconserved class of membrane proteins similar to bacterial transportersand involved in normal physiological transport processes. Sequenceanalysis of the mdrl gene indicates that Pgp consists of 1280 aminoacids distributed between two homologous (43% identity) halves. Eachhalf of the molecule has six hydrophobic transmembrane domains and eachhas an ATP binding site within the large cytoplasmic loops. Only about8% of the molecule is extracellular, and the carbohydrate moiety(approximately 30 kDa) is bound to sites in this region.

Thus, it will be appreciated that mammalian cells having a“multidrug-resistance” or “multidrug-resistant” phenotype arecharacterized by the ability to sequester, export or expel a pluralityof cytotoxic substances (e.g., chemotherapeutic drugs) from theintracellular milieu. Cells may acquire this phenotype as a result ofselection pressure imposed by exposure to a single chemotherapeutic drug(the selection toxin). Alternatively, cells may exhibit the phenotypeprior to toxin exposure, since the export of cytotoxic substances mayinvolve a mechanism in common with normal export of cellular secretionproducts, metabolites, and the like. Multidrug resistance differs fromsimple acquired resistance to the selection toxin in that the cellacquires competence to export additional cytotoxins (otherchemotherapeutic drugs) to which the cell was not previously exposed.For example, Mirski et al. (1987), 47 Cancer Res. 2594-2598, describethe isolation of a multidrug-resistant cell population by culturing theH69 cell line, derived from a human small cell lung carcinoma, in thepresence of adriamycin (doxorubicin) as a selection toxin. Survivingcells were found to resist the cytotoxic effects of anthracyclineanalogs (e.g., daunomycin, epirubicin, menogaril and mitoxantrone),acivicin, etoposide, gramicidin D, colchicine and Vinca-derivedalkaloids (vincristine and vinblastine) as well as of adriamycin.Similar selection culturing techniques can be applied to generateadditional multidrug-resistant cell populations. Accordingly, thepharmaceutical compositions of the invention can additionally includecompounds which act to inhibit the MDR phenotype and/or conditionsassociated with MDR phenotype. Such compounds can include any known MDRinhibitor compounds in the art, such as, antibodies specific for MDRcomponents (e.g. anti-MDR transporter antibodies) or small moleculeinhibitors of MDR transporters, including specifically, tamoxifen,verapamil and cyclosporin A, which are agents known to reverse orinhibit multidrug resistance. (Lavie et al. J. Biol. Chem. 271:19530-10536, 1996, incorporated herein by reference). Such compounds canbe found in U.S. Pat. Nos. 5,773,280, 6,225,325, and 5,403,574, each ofwhich are incorporated herein by reference. Such MDR inhibitor compoundscan be co-administered with the anti-mesothelin immunoconjugates of theinvention for various purposes, including, reversing the MDR phenotypefollowing the detection of the MDR phenotype to assist or enhance achemotherapeutic treatment. The MDR inhibitor, such as, for example,tamoxifen, verapamil or cyclosporin A, may be used in conjuction withthe compounds of the invention to assist in the detection of the MDRphenotype. In accordance with this aspect, an MDR inhibitor can enhancethe uptake and accumulation of a compound of the invention in an MDRcancer cell since the capacity of the MDR transport system intransporting or “pumping out” the imaging compound vis-a-vis thesubstrate domain would be diminished in the presence of an MDRinhibitor.

In yet another embodiment, the anti-mesothelin immunoconjugates of thepresent invention are used in association with a gene therapy program inthe treatment of cancer. Gene therapy with recombinant cells secretinginterleukin-2 can be administered in combination with the inventiveimmunoconjugates to prevent or treat cancer, particularly breast cancer(See, e.g., Deshrnukh et ah, 2001, J. Neurosurg. 94:287).

To assess the ability of a particular immunoconjugate to betherapeutically useful to treat cancer, as an example, theimmunoconjugate may be tested in vivo in a mouse xenograft tumor model.Examples of therapeutic models are detailed in Examples 1 and 2.Antibody activity may also be tested using an antibody dependentcell-mediated cytotoxicity assay as described in Example 3.

Pharmaceutical Compositions and Dosages

The immunoconjugates described herein may be provided in apharmaceutical composition comprising a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier may be non-pyrogenic.The compositions may be administered alone or in combination with atleast one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. A variety of aqueous carriers may be employed including, but notlimited to saline, glycine, or the like. These solutions are sterile andgenerally free of particulate matter. These solutions may be sterilizedby conventional, well-known sterilization techniques (e.g., filtration).

Generally, the phrase “pharmaceutically acceptable carrier” is artrecognized and includes a pharmaceutically acceptable material,composition or vehicle, suitable for administering compounds of thepresent invention to mammals. The carriers include liquid or solidfiller, diluent, excipient, solvent or encapsulating material, involvedin carrying or transporting the subject agent from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations. Wetting agents, emulsifiers and lubricants,such as sodium lauryl sulfate and magnesium stearate, as well ascoloring agents, release agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the immunoconjugate compositions of the invention.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, and the like. The concentration ofthe immunoconjugate of the invention in such pharmaceutical formulationmay vary widely, and may be selected primarily based on fluid volumes,viscosities, etc., according to the particular mode of administrationselected. If desired, more than one type of antibody or immunoconjugatemay be included in a pharmaceutical composition (e.g., an antibody withdifferent Ka for mesothelin binding).

The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones. In addition to theactive ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the active compounds intopreparations which may be used pharmaceutically. Pharmaceuticalcompositions of the invention may be administered by any number ofroutes including, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, parenteral,topical, sublingual, or rectal means.

The compositions of the invention additionally contemplate suitableimmunocarriers, such as, proteins, polypeptides or peptides such asalbumin, hemocyanin, thyroglobulin and derivatives thereof, particularlybovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH),polysaccharides, carbohydrates, polymers, and solid phases. Otherprotein-derived or non-protein derived substances are known to thoseskilled in the art.

In aspects involving vaccines, e.g. cancer vaccines together with theantibodies of the invention, the compositions of the invention can beadministered with or without an adjuvant. Administration can be carriedout in the absence of an adjuvant in order to avoid any adjuvant-inducedtoxicity. The person of ordinary skill in the art to which thisinvention pertains, e.g. a medical doctor specializing in cancer, willappreciate and understand how to ascertain whether an adjuvant should orshould not be used and can dependent upon the medical history of asubject, family data, toxicity data, allergy-related test results, etc.In embodiments where an adjuvant is used, it is advantageous that theadjuvant promotes the formation of protective antibodies, such asprotective IgG antibodies. Any suitable adjuvant known to one ofordinary skill in the art is contemplated by the present invention andare readily adapted to this invention. Suitable adjuvants for use invaccinating animals can include, but are not limited to, aluminumhydroxide, saponin-and-its purified component Quit A, complete Freund'sadjuvant (CFA) and incomplete Freund's adjuvant (IFA). Dextran sulfatehas been shown to be a potent stimulator of IgG2 antibody againststaphylococcal cell surface antigens, and also is suitable as anadjuvant. It will be appreciated by the skilled person that someadjuvants can be more preferable for veterinary application, whereasother adjuvants will be preferable for use in humans, and that adjuvanttoxicities are a consideration that should be made by the skilled personprior to administration of the compound to a human.

Formulations suitable for parenteral, subcutaneous, intravenous,intramuscular, and the like; suitable pharmaceutical carriers; andtechniques for formulation and administration may be prepared by any ofthe methods well known in the art (see, e.g., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 20th edition, 2000). Liquiddosage forms for oral administration of the compounds of the inventioninclude pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to the amount of an immunoconjugate that may be used toeffectively treat a disease (e.g., cancer) compared with the efficacythat is evident in the absence of the therapeutically effective dose.

The therapeutically effective dose may be estimated initially in animalmodels (e.g., rats, mice, rabbits, dogs, or pigs). The animal model mayalso be used to determine the appropriate concentration range and routeof administration. Such information may then be used to determine usefuldoses and routes for administration in humans. Therapeutic efficacy andtoxicity (e.g., ED50—the dose therapeutically effective in 50% of thepopulation and LD50—the dose lethal to 50% of the population) of animmunoconjugate may be determined by standard pharmaceutical proceduresin cell cultures or experimental animals. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it may be expressed asthe ratio, LD50/ED50. The data obtained from animal studies may used informulating a range of dosage for human use. The dosage contained insuch compositions may be within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

The exact dosage may be determined by the practitioner, in light offactors related to the patient who requires treatment. Dosage andadministration may be adjusted to provide sufficient levels of theimmunoconjugate or to maintain the desired effect. Factors that may betaken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Polynucleotidesencoding immunoconjugates of the invention may be constructed andintroduced into a cell either ex vivo or in vivo using well-establishedtechniques including, but not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and DEAF- orcalcium phosphate-mediated transfection.

Effective in vivo dosages of toxophore components of an immunoconjugateare in the range of about 5 μg to about 500 μg/kg of patient bodyweight. The mode of administration of immunoconjugate-containingpharmaceutical compositions of the present invention may be any suitableroute which delivers the antibody to the host. As an example,pharmaceutical compositions of the invention may be useful forparenteral administration (e.g., subcutaneous, intramuscular,intravenous, or intranasal administration). All patents and patentapplications cited in this disclosure are expressly incorporated hereinby reference. The above disclosure generally describes the presentinvention. A more complete understanding can be obtained by reference tothe following specific examples, which are provided for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES Example 1 Immunoconjugate Efficacy in a Mesothelin ExpressingHuman Pancreatic Carcinoma Xenograft Mouse Model

In order to analyse if the anti-mesothelin immunoconjugates were able toreduce the growth of tumors in a mesothelin dependent manner, humanpancreatic carcinoma cells (MiaPaCa-2) were stably transfected withmesothelin and used to establish a subcutaneously growing tumor mousemodel. The human colon carcinoma cell line HT29 was used to establishmesothelin negative control tumors within the efficacy study. MiaPaCacells were maintained as adherent cultures in DMEM medium supplementedwith 10% (v/v) FCS, 2.5% (v/v) horse serum, 1.5 g/l sodiumbicabonate,4.5 g/l glucose, 4 mM glutamine and 0.4% (v/v) Hygromycin. HT29 cellswere cultured in McCoy's 5a medium with 1.5 mM glutamine, 2.2 g/lsodiumbicarbonate and 10% (v/v) FCS. Mesothelin expression of MiaPaCa-2cells and absence of mesothelin in HT29 cells was confirmed by FACS (notshown). To assess in vivo growth of the tumor cells, female NMRI nudemice were subcutaneously inoculated into the right flank with 3×10⁶MiaPaCa-2 cells or 1×10⁶ HT29 cells, resuspended in 50% Matrigel™ and50% Medium. As anti-mesothelin immunoconjugates MF-J-SPDB-DM4,MF-T-SPDB-DM4, MF226-SPDB-DM4 and MOR6640-SPDB-DM4 have been tested attreatment doses of 0.01 mg/kg, 0.03 mg/kg, 0.05 mg/kg and 0.2 mg/kg(related to the amount of toxophore). MF-J-SPDB-DM4, MF-T-SPDB-DM4,MF226-SPDB-DM4 and MOR6640-SPDB-DM4 were generated by the followingprocedure: Anti-mesothelin antibodies were modified with4-[2-pyridyldithio]butanoic acid N-hydroxsuccinimide ester (SPDB) tointroduce dithiopyridyl groups. At 8 mg/mL antibody, a ˜6-fold molarexcess of SPDB (˜20 mM stock solution in EtOH) was used to modify theantibody. Modified antibodies were reacted with a 11-fold molar excessof the free thiol form of maytansinoid over thiopyridyl. The reactionwas carried out at 2.5 mg/nil antibody in the presence of 3%dimethylacetamide (3% v/v) for 20 hours at room temperature. Theconjugation reaction mixture was purified from unreacted drug andreaction byproducts using a desalting Sephadex G25 column. The number ofmaytansinoid molecules per antibody was calculated by measuringabsorbances at 252 nm and at 280 nm, using extinction coefficients of224000 M⁻¹ cm⁻¹ for antibody and 5180 M⁻¹ cm⁻¹ for DM4 at 280 nm. The252 nm/280 nm absorbance ratio is 0.37 for antibody and 5.05 for DM4.

Treatment started after tumor establishment at day 5 after tumor cellinoculation, followed by two further treatments on days 8 and 12 aftertumor cell inoculation. Control mice were either treated with 0.2 mg/kgof the non-targeting immunoconjugate (anti-lysozyme-SPDB-DM4) or withequal volumes of vehicle alone (10 mM histidine, 130 mM glycine, 5%(w/v) sucrose, pH 5.5). Treatments occurred with a dosage volume of 100μl/10 g body weight via intravenous application. Groups consisted of 6animals each. The health status of the mice was examined daily. Lengthand width of the subcutaneous tumors were measured using an electroniccaliper twice per week. Tumor area was calculated by the formula: tumorarea [mm²]=length [mm]×width [mm]. All data obtained throughout theexperiment were documented. An example of anti-tumor efficacy of antimesothelin immunoconjugate MF-T-SPDB-DM4 on mesothelin-transfected humanpancreas carcinoma cells at different treatment doses is shown inFIG. 1. Female NMRI nude mice were inoculated with 3×10⁶ mesothelinpositive MiaPaCa-2 human pancreas carcinoma cells (A) or 1×10⁶mesothelin negative HT29 human colon carcinoma cells (B) resuspended in50% Matrigel™/50% medium into their right flank. 5, 8, and 12 days aftertumor cell inoculation, mice received 0.01, 0.03, 0.05, 0.2 mg/kgMF-T-SPDB-DM4, (all concentrations relate to the amount of toxophore),or vehicle alone. Length and width of the tumors were measured twice perweak and tumor area was calculated by multiplication of width andlength. Mean values and standard derivation for every group andmeasuring time point are plotted. All n=6. Asterisks indicate P-values<0.05.

The treatment of the tumor-bearing mice revealed that allanti-mesothelin immunoconjugates tested were able to suppress growth ofthe mesothelin positive MiaPaCa-2 tumors in vivo at doses of 0.03 mg/kg,0.05 mg/kg and 0.2 mg/kg. At doses of 0.05 mg/kg and 0.2 mg/kg ofMF-T-SPDB-DM4 complete tumor eradication without regrowth of the tumorsoccured until the end of the observation period of 132 days. 0.05 mg/kgof the non-targeting control anti-lysozyme-SPDB-DM4 had no effect on themesothelin positive MiaPaCa tumor growth (Table 1). Compared tountreated and vehicle treated tumors, growth of mesothelin negative HT29tumors was not significantly reduced by the highest dose of 0.2 mg/kgMF-T-SPDB-DM4. This demonstrates that the strong tumor inhibitoryefficacy of MF-T-SPDB-DM4 is dependent on the expression of mesothelinwithin the tumor.

TABLE 1 Tumor inhibitory efficacy of anti-mesothelin immunconjugates inthe mesothelin positive MiaPaCa xenograft tumor model. MF- α MF-J- MOR-MF-T- 226- Lysozyme- DM4 6640-DM4 DM4 DM4 DM4 (control) DM4/Ab 2.8 3.63.6 3.3 4.0-4.1 0.01 mg/kg − − − − n.d. 0.03 mg/kg n.d. + + + n.d. 0.05mg/kg + + ++ a) + −  0.2 mg/kg + ++ b) ++ a) ++ + b) ++ tumoreradication + reduction/regression − no sign. Effect, Exp. duration20-30 d n.d. not determined a) Complete eradication over 132 d b) Tumorregrowth in all animals (n = 6) within 132 d

Example 2 Efficacy in Tumors Endogenously Expressing Mesothelin andComparison of Different Linkers

In order to test whether anti-mesothelin immunoconjugates are able tosuppress the growth of endogenously mesothelin expressing tumor cells invivo, a xenograft model with subcutaneously growing human cervixcarcinoma cells (HeLaMATU) was used. HeLaMATU cells were maintained asadherent cultures in DMEM/HAMS12 medium supplemented with 10% (v/v) FCS,2.5% (v/v) horse serum, 1% sodium pyruvate, and 1% (wiv) glutamine.Mesothelin expression was confirmed by FACS analysis in vitro. FemaleNMRI nude mice were subcutaneously inoculated with 1.5×10⁶ HeLaMATUcells resuspended in 50% Matrigel™/50% Medium, into the right flank.Additionally it was addressed whether exchange of the cleavableSPDB-linker by a polar (-sulfo-SPDB), by a stable linker (-SMCC), or bya polar and stable linker (-(PEG)4-mal) leads to an altered anti-tumorefficacy of the MOR6640-based immunoconjugate in vivo. HeLaMATUtumor-bearing mice were treated intravenously with 0.2 mg/kg of eitherMOR6640-SPDB-DM4, MOR6640-SMCC-DM1, MOR6640-Sulfo-SPDB-DM4, orMOR6640-(PEG)4-mal-DM1 (related to the amount of toxophore) at day 5, 8,and 12 after tumor cell inoculation. Control mice were either treatedwith 0.2 mg/kg of the non-targeting immunoconjugate(anti-lysozyme-SPDB-DM4) or with equal volumes of vehicle alone Groupsconsisted of 6 animals each. Daily examination of the health status ofthe mice was conducted. Length and width of the subcutaneous tumors weremeasured using an electronic caliper twice per week. Tumor area wascalculated by the formula: tumor area [mm²]=length [mm]×width [mm]. Dataobtained are presented in FIG. 2. Treatment of the tumor-bearing micerevealed a) that anti-mesothelin immunoconjugates were efficacious insuppressing growth of tumors expressing mesothelin endogenously in vivoand b) that conjugates with cleavable linkers (MOR6640-SPDB-DM4 andMOR6640) displayed a higher anti-tumor efficacy than the conjugates withstable linkers (MOR6640-SMCC-DM1 and MOR6640-(PEG)4-mal-DM1). Inparticular, MOR6640-SPDB-DM4 and MOR6640-sulfo-SPDB-DM4 led to aneradication of the tumors of all treated animals eleven days after thelast treatment, whereas treatment with MOR6640-SMCC-DM1 andMOR6640-(PFG)4-mal-DM1 resulted only in a delay of tumor growth.However, eleven days after the last treatment the area of tumors treatedwith MOR6640-SMCC-DM1 and MOR6640-(PEG)4-mal-DM1, respectively, weresignificantly smaller as compared to vehicle or anti-lysozyme-SPDB-DM4treated tumors. The non-targeting control conjugate had no effect ontumor growth. In order to compare the anti-tumor efficacy of thedifferent linkers in a second xenograft model, we employed the modelwith subcutaneously growing vector- or mesothelin-transfected (#37)MiaPaCa-2 cells (human pancreas carcinoma cells). MiaPaCa-2-vector andMiaPaCa-2#37 cells were maintained as adherent cultures in DMEM/HAMS12medium supplemented with 10% (v/v) FCS, 1% (w/v) glutamine, and 0.1 mMnon-essential amino acids. Mesothelin expression was confirmed by FACSanalysis and by immunohistochemical analysis of subcutaneous tumors exvivo. Female NMRI nude mice were subcutaneously inoculated with 3×10⁶MiaPaCa-2-vector and MiaPaCa-2#37 cells, resuspended in 50%Matrigel™/50% Medium, respectively, into the right flank. Tumor-bearingmice were treated with 0.05 mg/kg of either MOR6640-SPDB-DM4,MOR6640-SMCC-DM1, MOR6640-Sulfa-SPDB-DM4, or MOR6640-(PEG)4-mal-DM1(related to the amount of toxophore) at day 5, 8, and 12 after tumorcell inoculation. Control mice were either treated with 0.05 mg/kg ofthe non-targeting immunoconjugate (anti-lysozyme-SPDB-DM4) or with equalvolumes of vehicle alone. Groups consisted of 6 animals each. Dailyexamination of the health status of the mice was conducted. Length andwidth of the subcutaneous tumors were measured using an electroniccaliper twice per week. Tumor area was calculated by the formula: tumorarea [mm²]=length [mm]×width [mm]. Tumor growth data are presented inFIG. 3. In mice bearing mesothelin expressing tumors, treatment withMOR6640-SPDB-DM4 and MOR6640-sulfa-SPDB-DM4 led to an eradication of thetumors of all treated animals 12 days after the last treatment. However,re-growth of these tumors was obtained ten days later (FIG. 3A).Treatment with MOR6640-SMCC-DM1, MOR6640-(PEG)4-mal-DM1, andanti-lysozyme-SPDB-DM4 did not affect tumor growth significantly. Incontrast to mesothelin expressing tumors, none of the treatments led toan altered growth of the vector-transfected MiaPaCa-2 cells in vivo(FIG. 3B).

Example 3 In Vitro Cytotoxicity of Anti-Mesothelin Immunoconjugates

To assess cytotoxicity of anti-mesothelin immunoconjugates, differentmeseothelin expressing cell lines were grown to 80-90% confluence,trypsinized and counted. Cells were then seeded into 384-well flatbottom plates at 800 with 25 ul of volume per well in their growth mediafor all cell lines. Media only wells were set up for blank subtraction.At 24 hr post seeding, MF-J-SPDB-DM4 MF-226-SPDB-DM4 MOR6640-SPDP-DM4,MF226-SPDP-DM4 and anti-Lysozyme-SPDP-DM4 were dosed at the range of0.01 to 3 00 nM. Triplicates were set up for each dilution. Endpointmeasurements were performed at 96 hrs. Cell viability was assessed byWST-1 assay measurement (Roche Cat #1644807). IC₅₀ values are shown inTable 2. A dose-response curve demonstrating in vitro cytotoxicity ofMF-T-SPDP-DM4 on HelaMatu cells is shown in FIG. 4.

TABLE 2 nM IC₅₀ values of anti-mesothelin immunoconjugates on mesothelinexpressing cell lines MF- αLyso- MF-J- MF-T - 226 - zyme - SPDP-MOR06640 - SPDP- SPDP- SPDP-DM4 IC50 (nM) DM4 SPDP- DM4 DM4 DM4(control) MiaPaCa-2 1.4 0.36 1.3 21 >100 (Mesothelin+)MiaPaCa-2 >100 >100 >100 >100 >100 (Mesothelin−) HT29 C2 0.3 0.21 1.5 818 (Mesothelin+) HT29 V >50 >50 >50 >50 >50 (Mesothelin−) CHO A9* 0.351.2 1.0 1.3 >50 (Mesothelin+) CHO K1* >50 >50 >50 >50 >50 (Mesothelin−)DU145 5.2 5.1 9 10 — HCT116 9.0 20.7 17.2 26.5 33.1SW480 >50 >50 >50 >50 >50 MDAMB231 >50 >50 >50 >50 >50MCF10a >50 >50 >50 >50 >50 HelaMatu 0.2 0.6 0.45 0.9 0.4 OVCAR-3 0.91.07 2.8 14.8 >100 KD of Fab 9.2 0.19 16.3 58.3 (nM)

TABLE 3 Sequences of antibodies Antibody VH VL VH VL HCDR1 HCDR2 HCDR3LCDR1 LCDR2 LCDR3 Protein Protein Nucleotide Nucleotide SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID MF-J 1 4 7 10 1316 20 24 28 32 MOR 1 4 7 10 13 17 21 25 29 33 06640 MF-226 2 5 8 11 1418 22 26 30 34 MF-T 3 6 9 12 15 19 23 27 31 35

What is claimed is:
 1. A method for treating pancreatic cancer,comprising administering to a subject in need thereof an effectiveamount of an immunoconjugate, wherein the immunoconjugate comprises aDM4 maytansinoid and a human or humanized antibody or functionalfragment thereof comprising an antigen-binding site that is specific forMesothelin (SEQ ID NO:36), and wherein the antigen-binding sitecomprises a variable heavy chain comprising complementarity determiningregions (CDRs) corresponding to SEQ ID NOS 3, 6, and 9 and a variablelight chain comprising complementarity determining regions (CDRs)corresponding to SEQ ID NOS 12, 15, and 19, and wherein the DM4maytansinoid and the human or humanized antibody or functional fragmentthereof are linked by an SPDB linker, and wherein the effective amountis from 0.05 mg DM4 maytansinoid/kg body weight up to 0.2 mg DM4maytansinoid/kg body weight.
 2. The method according to claim 1, whereinthe immunoconjugate is administered together with one or moreanti-cancer agents to potentiate the effect of the immunoconjugate, theone or more anti-cancer agents, or both.
 3. The method according toclaim 2, wherein the anti-cancer agent is selected from the groupconsisting of gemcitabine, paclitaxel, docetaxel, cisplatin,carboplatin, doxorubicin, and liposomal doxorubicin.
 4. The methodaccording to claim 1, wherein the human or humanized antibody orfunctional fragment thereof comprises a variable heavy chain of SEQ IDNO: 23 and a variable light chain of SEQ ID NO: 27.